Ataxia Telangiectasia is a genomic instability syndrome in which mutations in the DNA damage checkpoint protein Atm are associated with cancer, infertility, immune dysfunction, and neurodegeneration. Atm plays a central role in cellular responses to double-stranded DNA breaks (DSB), while a second pathway involving the related checkpoint protein Atr responds to replication stress and bulky DNA lesions, as well as DSB. The extent of functional overlap between these pathways is not well understood, in part because components of the Atr pathway, which also includes Chk1 and the Rad9-Rad1-Hus1 (911) complex, are essential for viability. The long-term objectives of the research described in this application are to resolve the relationship between the Atm and Atr pathways, and to determine how the activity of the Atr pathway affects disease pathogenesis when Atm is defective. The investigators employed a Hus1 allelic series in mice to identify genetic interactions between the Atm and Atr pathways. Synthetic lethality was observed when partial Hus1 impairment was combined with Atm deficiency, establishing an essential cooperative relationship between the two primary mammalian DNA damage checkpoint pathways. Although a severe reduction in Hus1 expression was lethal in combination with Atm loss, a slightly higher level of Hus1 expression yielded viable mice at less than expected frequency. In Specific Aim 1, the basis for the embryonic lethality will be determined by morphological and histological analysis of embryos, as well as by examination of DNA damage signaling in cultured cells with both Atm and Hus1 defects. In addition, surviving mice with simultaneous Atm and Hus1 defects will be tested for neurodegeneration, a prominent phenotype of Ataxia Telangiectasia patients that is not observed in Atm knock-out mice. In Specific Aim 2, the impact of reduced Hus1 function on tumor development in Atm-deficient mice will be assessed. Atm heterozygosity confers an increased risk of breast cancer in humans, and therefore tumorigenesis will also be examined in heterozygous Atm mice with a partial Hus1 defect. Taken together, the proposed studies will clarify the relationship between two primary mammalian DNA damage checkpoint pathways and resolve the cooperative roles for these genome maintenance mechanisms in executing developmental programs, preventing neurodegeneration, and suppressing tumorigenesis.